Brain nitric oxide synthases in the

interleukin-1b-induced activation of

hypothalamic-pituitary-adrenal axis

Anna G¹dek-Michalska, Joanna Tadeusz, Paulina Rachwalska,

Jadwiga Spyrka, Jan Bugajski

Department of Physiology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków,Poland

Correspondence: Anna G¹dek-Michalska, e-mail: gadek@if-pan.krakow.pl

Abstract:

Background: Interleukin-1b (IL-1b), the major cytokine involved in activation of hypothalamic-pituitary-adrenal (HPA) axis

modulates both central and peripheral components regulating HPA activity. The role of nitric oxide (NO) generated by neuronal ni-

tric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in brain structures involved in HPA axis regulation has not

been elucidated. The aim of the study was to assess the receptor selectivity of IL-1b stimulatory action on HPA axis and to determine

the involvement of nNOS and iNOS in this stimulation.

Methods: Experiments were performed on male Wistar rats which were injected intraperitoneally (ip) with IL-1b (5 µg/kg) or IL-1

receptor antagonist (IL-1ra) (50 µg/kg or 100 µg/kg) 15 min before IL-1b. Rats were sacrificed by rapid decapitation 1, 2 or 3 h after

IL-1b administration. Trunk blood for ACTH, corticosterone and IL-1b determinations was collected and prefrontal cortex, hippo-

campus and hypothalamus were excised and snap frozen. Western blot analyses were performed and IL-1b, nNOS and iNOS protein

were determined in brain structures samples.

Results: IL-1b significantly increased plasma ACTH, corticosterone and IL-1b levels during 2 h after ip administration. IL-1 recep-

tor antagonist was able to abolish the stimulatory effect of IL-1b on plasma ACTH and corticosterone levels and significantly, but

not totally, reduced plasma IL-1b level. The role of NO in prefrontal cortex, hippocampus and hypothalamus in the IL-1b-induced

HPA axis activity alterations was determined by measuring the changes in nNOS and iNOS levels. The highest level of both izoen-

zymes 1 h following IL-1b administration decreased in a regular, parallel manner 2 and 3 h later, approaching control values. These

changes were almost totally prevented by pretreatment with IL-1 receptor antagonist. In the hypothalamus the IL-1b-induced initial

significant increase of nNOS regularly decreased in a modest rate and remained at significant higher level compared to control val-

ues. By contrast, iNOS level gradually increased 2 and 3 h after IL-1b administration in a significant time-dependent manner. The

changes in both NOS izoenzyme levels in hypothalamus were suppressed by pretreatment with IL-1 receptor antagonist. Results also

show that a regular and parallel decrease of nNOS in the hypothalamus and prefrontal cortex are parallel in time and magnitude to re-

spective fall in plasma IL-1b and ACTH levels.

Conclusion: The present study suggests that the IL-1b-induced transient stimulation of HPAaxis activity is parallel in time and mag-

nitude to the respective changes of nNOS in hypothalamus and prefrontal cortex, the brain structures involved in regulation of HPA

axis activity.

Key words:

interleukin-1b, nitric oxide synthases, interleukin-1 receptor antagonist, immuno-endocrine responses, limbic-hypothalamic-adrenal axis

Pharmacological Reports, 2012, 64, 1455�1465 1455

Pharmacological Reports2012, 64, 1455�1465ISSN 1734-1140

Copyright © 2012by Institute of PharmacologyPolish Academy of Sciences

Introduction

Cytokines secreted by cells of the immune system

may also be synthesized and secreted by non immune

cells and act as signal transmitters on neuroimmune

cells [2, 5, 6]. Interleukin-1b (IL-1b) is one of the ma-

jor cytokine involved in a stereotyped set of re-

sponses, including the regulation of HPA axis that is

mediated by the brain [16, 25]. Peripheral administra-

tion of IL-1b in rats activates the HPA axis resulting

in increased plasma levels of ACTH and glucocorti-

coids. IL-1b is expressed in all components of the

HPA axis and affects all its levels stimulating the se-

cretion of corticotrophin releasing hormone (CRH)

from the hypothalamus and ACTH stimulates the se-

cretion of glucocorticoids from adrenal cortex [10, 13,

21]. Furthermore, IL-1b also directly induces ACTH

secretion from the pituitary via IL-1 type I receptors

and also stimulates the secretion of corticosterone

from the adrenal, although IL-1 receptors were not

found in this gland [1, 7, 36].

Although hypothalamic CRH is considered a primary

mechanism by which cytokines stimulate HPA axis, evi-

dence indicates a direct action of cytokines at the level

of pituitary and adrenal glands. In addition to circulating

cytokines which may act upon all three levels of the

HPA axis, cytokines are generated in the brain, the ante-

rior pituitary and the adrenal cortex [2, 16].

Although the blood-brain barrier (BBB) presents

a physical barrier to cytokine-CNS interaction, they

may enter the brain 1) via fenestrated endothelium of

the circumventricular organs (CVOs) [8, 27] 2) via

limited active transport system, 3) induction of gen-

eration of cytokines from cells of the BBB, which

then secrete cytokines into the brain parenchyma [31,

32, 34] and 4) a transport across the BBB by infiltrat-

ing leukocytes. Cytokine may act at a distance by af-

fecting the CNS via noradrenergic, serotonergic, do-

paminergic or GABA-ergic pathways [20, 30, 39] and

stimulate ascending visceral peripheral neural path-

ways [29], mainly vagus nerves which convey signals

to endocrine neurons in the paraventricular nucleus

(PVN) and supraoptic nuclei (SON) of the hypothala-

mus [9]. Vagal stimulation significantly activates

magnocellular and parvocellular endocrine neurons

within the PVN [19] and SON in rats to the CRH neu-

rons within the PVN. Viscerosensory inputs to the

CRH neurons within the PVN are preferentially nora-

drenergic. In the hypothalamus the noradrenergic in-

nervation of the paraventricular nuclei has been

shown to mediate the IL-1 activation of the HPA axis.

Cytokine normally produced by peripheral immune

cells are also generated by CNS glial cells. Cytokine

can markedly affect neurotransmission within emo-

tion regulatory brain circuits. Neuroimmune interac-

tions involving peripheral and glial derived cytokines

play an important role in depression [17].

Nitric oxide (NO) as a signaling molecule among

various cell types and systems serves also as a neuro-

transmitter in the brain [20, 22, 23, 35]. It is synthe-

sized by nitric oxide synthase, neuronal (nNOS) and

inducible (iNOS). Under physiological conditions

nNOS is constitutively expressed, and it is the major

isoenzyme of NOS in the brain. Inductible NOS is un-

detectable under basal conditions and it is upregulated

in response to various stimuli such as inflammatory

cytokines and stress [4, 14, 24, 40]. NO generated by

iNOS in the brain is involved in central CRH-induced

elevation of plasma CRH, but it is not clear whether

nNOS plays a role in these CRH induced elevations.

Neuronal NOS is highly expressed by cells of the

hypothalamic PVN, where the sympatho-adrenal sys-

tem, the HPA axis and the hypothalamo-neurohypo-

physeal system originates. These structures regulate

the neuroendocrine stress responses. NO generated by

nNOS plays a significant role in modulating the activ-

ity of these systems under acute stressor exposure [26,

28]. NOS constitutive forms are found in PVN CRH

and AVP neurons as well as in direct and indirect af-

ferents to the PVN such as hippocampus, the dor-

somedial lateral part of the hypothalamus and amyg-

dale [33]. However, there is not always concordance

between the regions of the hippocampus or amygdale

that projects to the PVN.

Stimulation of hippocampus lowers glucorticoid

secretion in rats and humans, lesions of hippocampus

increase corticosterone release. Hippocampus damage

also increases PVN CRH and vasopressin gene ex-

pression and extent of PVN neural activation after

stress, suggesting that hippocampal inhibition of the

HPA is mediated via PVN [18].

Medial prefrontal cortex (MPC) is implicated in

HPA axis inhibition. Lesions of prelimbic and anterior

cingulated subregions of the MPC increases ACTH

and corticosterone secretion and PVN c-Fos mRNA

induction after restraint stress. The role of corticoster-

one in HPA integration may depend on subregion and

hemisphere.

1456 Pharmacological Reports, 2012, 64, 1455�1465

This study was designed to evaluate the HPA re-

sponse to a challenge dose of IL-1b 1, 2 and 3 h after

administration. Also the role of NO in prefrontal cortex,

hippocampus and hypothalamus in the IL-1b-induced

HPA axis activity alterations was assessed by measur-

ing the changes in nNOS and iNOS protein levels.

Materials and Methods

Animals

Adult male Wistar rats (190–220 g; Charles River

Laboratories, Sulzfeld, Germany) were used in all the

experiments. The animals were kept in groups of 5 per

cage (52 × 32 × 20 cm) under controlled conditions

(a 12/12 h light/dark cycle with the light on at 7:00 a.m.,

a room temperature of 22 ± 2°C), with free access to

standard laboratory food and tap water. Rats were

given 1-week acclimation period before experimental

session. All experiments were carried out in accor-

dance with bioethical requirements and protocols were

approved by the Local Bioethics Commission for

Animal Experiments at the Institute of Pharmacology,

Polish Academy of Sciences, Kraków, Poland.

Experimental procedure

Experiments were performed on 3 main groups of

rats: control animals injected intraperitoneally (ip)

with saline, animals injected ip with a single dose of

IL-1b (5 µg/kg, Sigma-Aldrich) and animals injected

ip with a dose of IL-1 receptor antagonist (50 µg/kg

or 100 µg/kg, Sigma-Aldrich) followed by IL-1b ad-

ministration 15 min later. Rats were sacrificed by

rapid decapitation 1 h, 2 h or 3 h after IL-1b admini-

stration. Trunk blood for plasma corticosterone and

IL-1b determinations was collected in the presence of

EDTA (2.5 mg/ml of blood, Sigma-Aldrich) or for

ACTH immunoassay in the presence of EDTA and

aprotinin (0.6 TIU/ml of blood; Sigma-Aldrich) and

centrifuged in 4°C within 30 min after collection.

After decapitation, the brain was rapidly removed

from the skull and prefrontal cortex, hippocampus

and hypothalamus were excised on ice-cold glass

plates. The tissues were immediately frozen on dry

ice and stored at –70°C. Protein extracts were pre-

pared according to G¹dek-Michalska et al. [15]. Total

corticosterone, ACTH and IL-1b levels were measured

in plasma using commercially available Rat/Mouse

Corticosterone EIA kit (Immunodiagnostic Systems),

ACTH Rat/Mouse EIA kit (Phoenix Pharmaceuticals)

and Rat IL-1b Platinum ELISA kit (eBioscience).

Western blot

Denaturated proteins (10 µg per lane) were separated us-

ing a 7.5% (for nNOS, iNOS) or 15% (for IL-1b) SDS-

polyacrylamide gel electophoresis (Mini PROTEAN®

Tetra Cell, Bio-Rad) under constant voltage (90 V in

stacking gel; 150 V in resolving gel), and were trans-

ferred electrophoretically (90 V, 1 h) onto a nitrocellu-

lose membrane (0.45 µm, Bio-Rad). The membranes

were NOS2 polyclonal antibody in dilution 1:400

(Santa Cruz Biotechnology), and to determine IL-1b

(17.5 kDa) level we used rabbit anti-IL-1b polyclonal

antibody in dilution 1:750 (Thermo Scientific). b-

Actin levels were determined with rabbit anti-b-actin

polyclonal antibody (1:400, Santa Cruz Biotechnol-

ogy) and used for normalization of nNOS, iNOS and

IL-1b bands. After incubation, the membranes were

washed four times for 10 min with TBST (TBS-0.1%

Tween 20) and finally incubated with goat anti-rabbit

horseradish peroxidase-conjugated secondary anti-

body (1 : 10,000, Santa Cruz Biotechnology) in 1%

non-fat dry milk in TBS-0.05% Tween 20 for 1 h at

room temperature. Afterwards, the membranes were

washed four times for 15 min with TBST, and the pro-

teins were detected using Immun-Star HRP Chemilumi-

nescence Kit (Bio-Rad) and visualized by a sensitive

CCD camera system (FujiLas 1000 Imager). The optical

density of appropriate bands was quantified by densi-

tometric analysis of blots using Image Gauge V4.0 Soft-

ware (Fujifilm) and normalized to b-actin levels. All the

values are expressed as a percentage of controls.

Statistical analysis

All results are expressed as the means ± standard error

of the mean (SEM) where n = 6–8 rats per group. Sta-

tistical analyses were performed using GraphPad

Prism 5 (GraphPad Software Inc.). Data were ana-

lyzed by one-way analysis of variance (ANOVA) fol-

lowed by Tukey’s multiple range test. Groups that are

significantly different from control are indicated in

the figures as: + p < 0.05, ++ p < 0.01, +++ p < 0.001,

and significant difference from IL-1b injected group

Pharmacological Reports, 2012, 64, 1455�1465 1457

Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.

is shown as: * p < 0.05, ** p < 0.01, *** p < 0.001 for

1 h time point and ^ p < 0.05, ^^ p < 0.01, ^^^ p <

0.001 for 2 h.

Results

Plasma IL-1b, ACTH and corticosterone

response to systemic IL-1b administration

Exogenous IL-1b (5 µg/kg) administered ip induced

significant increase its plasma level much stronger

after 1 h than after 2 h. This increase was IL-1b selec-

tive, since it was reduced by ip injection of IL-1 re-

ceptor antagonist (IL-1ra) 15 min before IL-1b. This

reduction was dose dependent, stronger after pretreat-

ment with 100 µg/kg than 50 µg/kg of IL-1ra (Fig.

1A). Exogenous IL-1b also significantly increased

plasma ACTH levels 1 and 2 h after administration.

This increase was also abolished by pretreatment with

both doses of IL-1ra 1 h and 2 h after cytokine ad-

ministration (Fig. 1B). IL-1b-induced considerable

increase of plasma corticosterone level which was to-

tally prevented by IL-1ra 2 h following IL-1b injec-

tion (Fig. 1C). These results suggest that the above

changes in plasma IL-1b, ACTH and corticosterone

levels are in a major part IL-1b receptor specific.

Effect of IL-1ra on IL-1b-induced nNOS and

iNOS levels in brain structures

IL-1b (5 µg/kg ip)-induced increases of nNOS and

iNOS levels in prefrontal cortex 1 h after IL-1b admini-

stration gradually declined 3 h following ip injection

(Fig. 2A). The IL-1b-induced significant increase of

nNOS level in prefrontal cortex 1 and 2 h after admini-

stration was almost totally abolished by pretreatment

with IL-1ra (Fig. 2B). Likewise, a marked ip IL-1b-

induced increase of iNOS in prefrontal cortex was

totally abolished by pretreatment with IL-1ra (Fig. 2C).

In the hippocampus IL-1b gradually increased

nNOS level significantly 3 h after its administration

and respective alterations of iNOS levels did not at-

tained the level of significance. (Fig. 3A). IL-1ra sub-

stantially impaired the IL-1b-induced nNOS level 1 h

after its administration and slightly diminished iNOS

level after 1 and 2 h (Fig. 3B, 3C).

In the hypothalamus 1 h following IL-1b adminis-

tration, nNOS reached its highest level (94%) which

gradually decreased 2 and 3 h later and remained at

significantly higher level (68%) compared with saline

control values. Conversely, iNOS level markedly in-

creased 1–3 h after IL-1b administration compared

with saline treated group (Fig. 4A).

1458 Pharmacological Reports, 2012, 64, 1455�1465

Fig. 1. Effect of interleukin-1 receptor antagonist (IL-1ra) onincreased plasma levels of interleukin-1b (IL-1b) (A), ACTH (B) andcorticosterone (C) induced by IL-1b administration. In Figures 1�4rats were injected intraperitoneally (ip) with IL-1ra (50 or 100 µg/kg)15 min before IL-1b ip (5 µg/kg) and decapitated 1, 2 and 3 h later. InFigures 1�6, 8�10 rats per group were used. + p < 0.05, ++ p < 0.01and +++ p < 0.001 vs. saline control group; * p < 0.05, ** p < 0.01 and*** p < 0.001 vs. IL-1b ip 1 h; ^p < 0.05 and^^^p < 0.001 vs. IL-1b ip 2 h

The IL-1b-induced significant increase of nNOS

levels in the hypothalamus was totally abolished by ip

pretreatment with IL-1ra 15 min earlier (Fig. 4B).

Likewise, a marked but somewhat weaker rise of

iNOS 1 h after IL-1b administration was abolished by

IL-1ra after 2 h (Fig. 4C). These results suggest that

the ip IL-1b-induced effects on nNOS and iNOS levels

in examined brain structures, potentially involved in

HPA axis regulation are IL-1b receptor selective.

IL-1b levels in brain structures induced

by exogenous IL-1b administration

Interleukin-1b (5 µg/kg) 1 h after administration con-

siderably and to a similar extent increased IL-1b level

in prefrontal cortex and hippocampus (187 and 192%,

respectively), relative to control values in saline

treated rats. Also marked increase of IL-1b content

appeared in hypothalamus. These increased IL-1b

levels in all three structures sharply diminished to the

lowest levels 2 h after ip IL-1b administration. In the

third hour, IL-1b levels in prefrontal cortex and hypo-

thalamus returned to their high values. Despite these

changes, IL-1b levels in examined brain structures re-

mained significantly higher 1–3 h following exoge-

nous IL-1b administration compared to control levels

(Fig. 5A–C).

NOS levels in brain structures following IL-1b

administration

Interleukin-1b (ip, 5 µg/kg) 1 h after administration

induced a significant increase in nNOS levels, most

pronounced in hypothalamus (92%) and in prefrontal

Pharmacological Reports, 2012, 64, 1455�1465 1459

Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.

Fig. 2. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in prefrontal cortex (A).Effect of IL-1ra (50 µg/kg or 100 µg/kg) on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after IL-1b (5 µg/kg) ip administration. Repre-sentative immunoblot showing the expression of nNOS and iNOS in prefrontal cortex (D). For other details see legend to Figure 1

CORTEX

cortex (51%). This increase gradually dropped in sub-

sequent 2 and 3 h with moderately faster rate in pre-

frontal cortex than in hypothalamus. By contrast, in

hippocampus nNOS level only moderately increased

2 and 3 h after IL-1b administration compared with

control level (Fig. 2A, 3A, 4A). IL-1b administered

systemically also increased iNOS levels in prefrontal

cortex and in hypothalamus 1 h after ip injection com-

pared to respective control levels. This increased

iNOS level in prefrontal cortex sharply declined 2 and

3 h after IL-1b treatment to nearly control level, while

the respective increase of iNOS in hypothalamus fur-

ther linearly raised compared with control level. The

ip IL-1b-induced alterations of iNOS level in hippo-

campus, like these of nNOS, were not significant

compared to control levels (Fig. 2A, 3A, 4A).

NOS and IL-1b in brain structures compared

with plasma IL-1 b levels

The ip IL-1b induced changes in nNOS, iNOS and

IL-1b levels in brain structures were compared with

the parallel plasma IL-1b levels in order to determine

possible central and systemic significance of IL-1b

1460 Pharmacological Reports, 2012, 64, 1455�1465

Fig. 3. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in hippocampus (A). Effectof IL-1ra on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after ip IL-1b. Representative immunoblot showing the expression of nNOSand iNOS in hippocampus (D). For other details see legend to Figure 1

HIPPOCAMPUS

and nitric oxide synthases in the HPA axis activity.

One hour after ip administration, IL-1b considerably

increased its plasma levels which gradually decreased

2 and 3 h after injection but persisted at significant

higher level compared with control plasma levels in

saline-treated rats. These changes in plasma IL-1b

levels were generally parallel in time but at relatively

much higher levels compared with respective signifi-

cantly lower alterations of IL-1b levels in the prefrontal

cortex, hippocampus and hypothalamus (Fig. 5A–C).

These results suggest similar pattern in transient altera-

tions of IL-1b levels in plasma and brain structures .

Changes in nNOS and iNOS in brain structures

in relation to plasma IL-1b, ACTH and

corticosterone levels

Systemic IL-1b considerably increased its plasma

level parallel with ACTH level. These levels were

most increased 1 h after administration and gradually

diminished in a regular manner in the following 2 h

(Fig. 6A). The IL-1b- induced increased nNOS levels,

higher in hypothalamus than in prefrontal cortex and

iNOS in prefrontal cortex also regularly declined in

a parallel manner to the levels of plasma IL-1b and

Pharmacological Reports, 2012, 64, 1455�1465 1461

Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.

Fig. 4. Influence of IL-1b ip on neuronal (nNOS) and inducible (iNOS) nitric oxide synthase content (in % of control) in hypothalamus (A). Effectof IL-1ra on IL-1b-induced nNOS (B) and iNOS (C) level 1 and 2 h after ip IL-1b. Representative immunoblot showing the expression of nNOSand iNOS in hypothalamus (D). For other details see legend to Figure 1

HYPOTHALAMUS

ACTH levels (Fig. 6B). These results suggest that

both nNOS and iNOS in prefrontal cortex and hypo-

thalamus may be involved in the IL-1b-induced acti-

vation of the HPA axis under basal conditions.

Discussion

In the present study, systemic administration of IL-1b

increased considerably plasma levels of IL-1b like

ACTH and corticosterone. IL-1b administered ip can

stimulate the CNS and HPA axis by direct activation

of the parts of HPA axis outside the blood-brain bar-

1462 Pharmacological Reports, 2012, 64, 1455�1465

Fig. 5. Changes in IL-1b, nNOS and iNOS content in prefrontal cortex(A), hippocampus (B) and hypothalamus (C) in relation to plasmaIL-1b level. For other details see legend to Figure 1

Fig. 6. Comparison of IL-1b-induced changes in plasma IL-1b, ACTHand corticosterone levels (A) with nNOS and iNOS content in hypo-thalamus and prefrontal cortex (B). For other details see legend toFigure 1

rier, the median eminence to release CRH from end-

ings of PVN neurons, and stimulation anterior pitui-

tary adrenocorticotroph cells to release ACTH or di-

rect stimulation of adrenal cortex [1]. Systemically

administered IL-1b also induces the IL-1b expression

and may create an autocrine signaling loop that influ-

ences the adrenal response and may explain the disso-

ciation between plasma ACTH and corticosterone

levels [3, 11]. The strong inhibition of these IL-1b-i-

nduced stimulations by ip pretreatment with IL-1 re-

ceptor antagonist in the present experiment indicates

that these effects depend, at least in a major part, on

IL-1 receptor stimulation.

Exogenous IL-1b may also reach central cytokine

receptors in brain structures involved in HPA axis

regulation [27]. IL-1b may trigger increased synthesis

and release of IL-1b second messengers, PGs and NO

from endothelial cells vs. perivascular cells of BBB,

which have complex interaction in regulating the tim-

ing and types of brain responses [34]. In the present

experiment, IL-1b induced considerably higher in-

crease in plasma corticosterone levels 2 h after ad-

ministration compared with also significant response

1 h after injection.

IL-1b, 1 h after ip administration, significantly in-

creased the IL-1b content in prefrontal cortex and hip-

pocampus to a similar extent and moderately aug-

mented its level in hypothalamus. These increased

levels markedly declined 2 h after IL-1b administra-

tion and were parallel but at considerably lower level

compared to the changes in plasma IL-1b levels. Under

basal conditions the rate of alterations in IL-1b con-

tent in brain structures involved in HPA axis regula-

tion seem to be structure and time dependent.

The present investigation suggests the involvement of

NO generated by nNOS and iNOS in prefrontal cortex,

hypothalamus and hippocampus in the ip IL-1b-in-

duced stimulation HPA activity during 1–3 h after ad-

ministration. Our results show that the effects of IL-1b

on nNOS and iNOS activity were almost totally pre-

vented by prior administration of IL-1ra, suggesting

that they are IL-1 specific. Our former investigations

indicated that NO plays essential role in the IL-1b-in-

duced HPA axis stimulation. L-NAME a general NOS

inhibitor considerably reduced the ACTH and corti-

costerone response to ip IL-1b in rats [14]. Repeated

stress markedly increased IL-1b generation in brain

structures involved in HPA axis regulation [15].

IL-1b induced the strongest and persistent increase

of nNOS protein level in the hypothalamus 1–3 h after

administration and a modestly weaker stimulatory ef-

fect in the prefrontal cortex. Conversely, in the hippo-

campus nNOS level increased in a linear manner 2–3 h

after IL-1b administration, which may be connected

with a comparably weaker and delayed action of the

hippocampus on HPA axis activity. In agreement with

our data, NO donor 3-morpholino-syndomine (SIN-1)

microinfused into the PVN, which contains majority

of the CRH neurons regulating HPA axis activity, sig-

nificantly increased plasma ACTH levels [33]. NO

generated in parvocellular neurons in the PVN stimu-

lates CRH and ACTH release [12, 33]. Microinfusion

into the hippocampus also increased plasma ACTH

levels to a smaller extent and a delayed onset com-

pared to that after PVN treatment. In our experiment

IL-1b considerably increased nNOS level in prefron-

tal cortex 1–2 h after administration. Increased NO

level in that structure is parallel with and may be in-

volved in enhanced HPA axis activity.

However, microinfusion of SIN-1 into the prefrontal

cortex was without effect. These data may suggest that

the NO may stimulate HPA axis when applied to different

brain regions. NOS izoenzymes also mediate centrally

CRH-induced sympathetic activation under stressful con-

ditions and may affect the HPA axis activity [37, 38].

Our present data suggest that the IL-1b-induced central

influence of NO on the HPA axis is stimulatory. They also

provide information regarding the role of hypothalamus

and prefrontal cortex in modulating the activity of NOS on

the HPA activity during stimulation with systemic IL-1b.

In the present experiment, the IL-1b-induced

changes of IL-1b levels in plasma were parallel in time

but relatively much larger than respective alterations in

prefrontal cortex, hippocampus and hypothalamus.

Also the alterations of IL-1b levels in plasma were

generally parallel with plasma ACTH and corticoster-

one levels. This suggests that IL-1b both in brain

structures and plasma is involved in the regulation of

HPA axis activity in non-stressed rats.

Acknowledgment:

This research was supported by grant: POIG 01.01.02-12-004/09-00financed by European Regional Development Fund.

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Received: May 22, 2012; in the revised form: September 19, 2012;accepted: September 25, 2012.

Pharmacological Reports, 2012, 64, 1455�1465 1465

Interleukin-1b, IL-1 receptor antagonist, nNOS, iNOS, HPA axisAnna G¹dek-Michalska et al.